1. Field of the Invention.
The invention relates to the detection of genetic abnormalities that confer. susceptibility to certain cancers in humans. More specifically, the invention relates to methods for detecting deletions of, or polymorphisms in, a newly discovered gene which encodes a tumor suppressor.
2. History of the Prior Art.
In recent years, a growing body of evidence has developed which supports the theory that the development of certain tumors is suppressed by gene products (xe2x80x9ctumor suppressorsxe2x80x9d) which inhibit cellular proliferation (see, e.g., the review in Marx, Science, 263:319-320, 1994). Conversely, if the tumor suppressors which would ordinarily be present in a cell are either absent (due, for example, to a gene deletion) or less active (due, for example, to a gene mutation), tumor growth which would otherwise be inhibited may go unchecked. However, although the growth of certain tumors has been positively demonstrated to relate to the deletion of a tumor suppressor expressing gene, it has not yet been shown that mutations in the same genes will allow abnormal cellular proliferation to occur.
The growth cycle of eukaryotic cells is regulated by a family of protein kinases known as the cyclin-dependent kinases (xe2x80x9cCDK""sxe2x80x9d). As shown in FIG. 1, the cyclins and their associated CDK""s move cells through the three phases of the growth cycle (G1, S and G2, respectively) leading to division in the mitosis phase (M). The cyclin/CDK complexes whose role in cellular proliferation has been most clearly defined to date are the cyclin D/CDK enzymes, which are believed to assist in the progression of the G1 growth cycle phase. Of these enzymes, cyclin D1 is believed to be an oncogene, whose overexpression stimulates excessive cell division through the continuous production of kinase, thus contributing to the development of cancers of, for example, the breast and esophagus. Cyclin D1 is specifically bound by CDK4 as part of a multi-protein complex that also consists of a protein known as p21 and cell nuclear antigen.
Known inhibitors of such cyclin/CDK overexpression include the tumor suppressor protein p53 and the protein product of the retinoblastoma (Rb) gene. Recently, another putative inhibitor (p16) was isolated and a cDNA for the inhibitor was partially sequenced by Serrano, et al., Nature, 366:704-710, 1993. The authors demonstrated that p16 binds CDK4 to inhibit the activity of the CDK4/cyclin D enzymes. Based on data indicating that p16 prevented phosphorylation by CDK/cyclin D of certain Rb growth cycle proteins, the authors proposed that p16 acts in vivo upstream and downstream of Rb to form a negative feedback loop to regulate cellular proliferation. However, no connection between p16 and the occurrence or inhibition of particular cancers was suggested, nor has any information been published concerning the genomic structure of the gene encoding p16.
Prior to the publication of the Serrano, et al., article referred to above, the inventors discovered a tumor suppressor gene (hereafter, xe2x80x9cCDK4Ixe2x80x9d) and identified its genomic structure (see, SEQ ID NO""s: 1-2). In non-malignant cells, CDK41 maps to chromosome 9p21 and is physically adjacent to the gene for methylthioadenosine phosphorylase (MTAse) (see, FIG. 4(b)). MTAse deficiencies resulting from deletions of, or mutations in, the gene for MTAse have been shown to be directly related to the onset of certain cancers (see, Nobori, etal., Cancer Res. 53:1098-1101, 1993, the disclosure of which are incorporated herein for reference regarding the role of MTAse in cancer development, and SEQ ID NO: 14, the nucleotide sequence of genomic MTAse).
Approximately one-half of all tumor cells which have been identified to date as either lacking CDK4I or containing mutations or rearrangements (collectively, xe2x80x9cpolymorphismsxe2x80x9d) of the CDK4I gene also lack MTAse. The inventors have also identified mutations in the CDK4I gene which are present in the tumor cells of patients with certain cancers. The invention is therefore directed to methods to detect (a) deletions of the CDK4I gene in cells, and (b) polymorphisms, which deletions and polymorphisms are indicative of susceptibility to certain cancers.
More specifically, in one aspect, the invention comprises methods for detecting point mutations in, or deletions of, the CDK4I gene. Such methods include polymerase chain reaction (PCR) based assays, gel electrophoresis of single-strand conformation polymorphisms, direct sequencing, and restriction endonuclease digestion. Detection of a deletion of the CDK4I gene will preferably be performed by a unique competitive PCR technique.
In another aspect, the invention comprises methods for detection of CDK4I proteins and biologically active fragments thereof (collectively, xe2x80x9cCDK4Ixe2x80x9d) in a biological cell sample.
In another aspect, the invention comprises screening protocols for susceptibility to particular cancers based on detection of polymorphisms associated with the occurrence of the cancers.
In another aspect, the invention comprises screening protocols for susceptibility to particular cancers based on detection of polymorphisms in, or deletions of, the genes for both CDK4I and MTAse, as well as detection of deficiencies in the products of the genes.
In another aspect, the invention comprises genomic CDK4I, expression products of the CDK4I gene, CDK4I and fragments thereof, as well as antibodies which will specifically bind CDK4I gene expression products, CDK41 and CDK4I fragments.